Development of rivaroxaban microemulsion-based hydrogel for transdermal treatment and prevention of venous thromboembolism.

We have developed a microemulsion (ME)-based hydrogel, containing propylene glycol, Azone®, Labrasol®, isobutanol and water (20:3:18:3:56), for the transdermal delivery of rivaroxaban (RVX). Formulation ME-1:RVX, which was loaded with 0.3 mg/g of RVX, presented as a clear, homogenous fluid with a droplet size of 82.01 ± 6.32 nm and a PdI of 0.207 ± 0.01. To provide gelation properties, 20 % (w/w) of Pluronic® F-127 was added to ME-1:RVX to generate formulation PME-1a. An added benefit was an increased capacity for RVX to 0.4 mg/g (formulation PME-1b). PME-1b displayed spherical droplets with a nanoscale diameter as observed by Transmission Electron Microscopy. The release of RVX from PME-1b was 20.71 ± 0.76 µg/cm2 with a permeation through pig epidermis of 18.32 ± 8.87 µg/cm2 as measured in a Franz Cell for 24 h. PME-1b presented a pseudoplastic behavior, pH value compatible with the skin and good stability over 60 days at room and elevated temperatures. The prothrombin time was assessed for each concentration of RVX obtained in the permeation assay and each demonstrated a relevant anticoagulant activity. PME-1b also presented no cytotoxicity against HaCaT cells. Utilizing GastroPlus® software, an in silico analysis was performed to simulate the delivery of PME-1b through a transdermal system that suggested a minimum dose of RVX for the treatment and prevention of venous thromboembolism could be achieved with an 8 h administration regimen. These results suggest that PME-1b is a promising transdermal formulation for the effective delivery of RVX that could be a viable alternative for the treatment and prevention of venous thromboembolism.

An attempt to chemically state the cross-talk between monomers of COX homodimers by double/hybrid inhibitors mofezolac-spacer-mofezolac and mofezolac-spacer-arachidonic acid.

Cardiovascular diseases (CVDs) account for over 17 million death globally each year, including arterial thrombosis. Platelets are key components in the pathogenesis of this disease and modulating their activity is an effective strategy to treat such thrombotic events. Cyclooxygenase-1 (COX-1) isoenzyme is involved in platelet activation and is the main target of non-steroidal anti-inflammatory drugs (NSAIDs) and new selective inhibitor research. Inhibitors of general formula mofezolac-spacer-mofezolac (mof-spacer-mof) and mofezolac-spacer-arachidonic acid (mof-spacer-AA) were projected to investigate the possible cross-talk between the two monomers (Eallo and Ecat) forming the COX-1 homodimer. Mofezolac was chosen as either one or two moieties of these molecules being the known most potent and selective COX-1 inhibitor and administrated to humans as Disopain™, then arachidonic acid (AA) was used to develop molecules bearing, in the same compound, in addition to the inhibitor moiety (mofezolac) also the natural COX substrate. Depending on the nature of the spacer, COX-1 and COX-2 activity was differently inhibited by mof-spacer-mof set with a preferential COX-1 inhibition. The highest COX-1 selectivity was exhibited by the compound in which the spacer was the benzidine [N,N'-(biphenyl-4,4'-di-yl)bis (2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamide) (15): COX-1 IC50 = 0.08 µM, COX-2 IC50 > 50 µM, Selectivity Index (SI) > 625]. In the case of mof-spacer-AA set, the COX inhibitory potency and also the isoform preference changed. (5Z, 8Z, 11Z, 14Z)-N-(4-{2-[3,4-Bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}butyl)icosa-5,8,11,14-tetraenamide (19) and (5Z, 8Z, 11Z, 14Z)-N-(4'-{2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}-[1,1'-biphenyl]-4-yl)icosa-5,8,11,14-tetraenamide (21), in which the spacer is the 1,2-diaminobutane or benzidine, respectively, selectively inhibited the COX-2, whereas when the spacer is the 1,4-phenylendiamine [(5Z, 8Z, 11Z, 14Z)-N-(4-{2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}phenyl)icosa-5,8,11,14-tetraenamide) (20) the COX preference is COX-1 (COX-1 IC50 = 0.05 µM, COX-2 IC50 > 50 µM, with a COX-1 selectivity > 1000). Molecular modelling by using FLAP algorithm shows fundamental interactions of the novel compounds at the entry channel of COX and inside its catalytic cavity. The effect of these mof-spacer-mof and mof-spacer-AA in inhibiting in vitro free arachidonic acid-induced platelet aggregation was also determined. A positive profile of hemocompatibility in relation to their influence on the blood coagulation cascade and erythrocyte toxicity was observed. Cytotoxicity and genotoxicity safety were also found for these two novel sets of compounds.

Nanoparticles Loaded with a New Thiourea Derivative: Development and In vitro Evaluation Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 µM for promastigotes and an IC50 of 70 µM for amastigotes. OBJECTIVE: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems. METHODS: Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM). RESULTS: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite Selectivity Index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). CONCLUSION: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

Vancomycin-loaded nanoparticles against vancomycin intermediate and methicillin resistant Staphylococcus aureus strains.

Bacterial infections represent one of the leading causes of mortality in the world. Among causative pathogens, S. aureus is prominently known as the underlying cause of many multidrug resistant infections that are often treated with the first-line choice antibiotic vancomycin (VCM). Loading antibiotics into polymeric nanoparticles (Np) displays promise as an alternative method to deliver therapy due to the greater access and accumulation of the antibiotic at the site of the infection as well as reducing toxicity, irritation and degradation. The aim of this work was to prepare, characterize and evaluate VCM-loaded nanoparticles (VNp) for use against S. aureus strains. Moreover, conjugation of Nps with holo-transferrin (h-Tf) was investigated as an approach for improving targeted drug delivery. VNp were prepared by double emulsion solvent evaporation method using PLGA and PVA or DMAB as surfactants. The particles were characterized for size distribution, Zeta Potential, morphology by transmission electron microscopy, encapsulation yield and protein conjugation efficiency. Process yield and drug loading were also investigated along with an in vitro evaluation of VNp antimicrobial effects against S. aureus strains. Results showed that Np were spontaneously formed with a mean diameter lower than 300 nm in a narrow size distribution that presented a spherical shape. The bioconjugation with h-Tf did not appear to increase the antimicrobial effect of VNp. However, non-bioconjugated Np presented a minimal inhibitory concentration lower than free VCM against a MRSA (Methicillin-resistant S. aureus) strain, and slightly higher against a VISA (VCM intermediate S. aureus) strain. VNp without h-Tf showed potential to assist in the development of new therapies against S. aureus infections.

Oral pentamidine-loaded poly(d,l-lactic-co-glycolic) acid nanoparticles: an alternative approach for leishmaniasis treatment.

Leishmaniasis is a group of diseases caused by a protozoa parasite from one of over 20 Leishmania species. Depending on the tissues infected, these diseases are classified as cutaneous, mucocutaneous and visceral leishmaniasis. For the treatment of leishmaniasis refractory to antimony-based drugs, pentamidine (PTM) is a molecule of great interest. However, PTM displays poor bioavailability through oral routes due to its two strongly basic amidine moieties, which restricts its administration by a parenteral route and limits its clinical use. Among various approaches, nanotechnology-based drug delivery systems (nano-DDS) have potential to overcome the challenges associated with PTM oral administration. Here, we present the development of PTM-loaded PLGA nanoparticles (NPs) with a focus on the characterization of their physicochemical properties and potential application as an oral treatment of leishmaniasis. NPs were prepared by a double emulsion methodology. The physicochemical properties were characterized through the mean particle size, polydispersity index (PdI), zeta potential, entrapment efficiency, yield process, drug loading, morphology, in vitro drug release and in vivo pharmacological activity. The PTM-loaded PLGA NPs presented with a size of 263 ± 5 nm (PdI = 0.17 ± 0.02), an almost neutral charge (-3.2 ± 0.8 mV) and an efficiency for PTM entrapment of 91.5%. The release profile, based on PTM dissolution, could be best described by a zero-order model, followed by a drug diffusion profile that fit to the Higuchi model. In addition, in vivo assay showed the efficacy of orally given PTM-loaded PLGA NPs (0.4 mg kg-1) in infected BALB/c mice, with significant reduction of organ weight and parasite load in spleen (p-value < 0.05). This work successfully reported the oral use of PTM-loaded NPs, with a high potential for the treatment of visceral leishmaniasis, opening a new perspective to utilization of this drug in clinical practice.

Development and Characterization of Dapsone Cocrystal Prepared by Scalable Production Methods.

In this study, the formation of caffeine/dapsone (CAF/DAP) cocrystals by scalable production methods, such as liquid-assisted grinding (LAG) and spray drying, was investigated in the context of the potential use of processed cocrystal powder for pulmonary delivery. A CAF/DAP cocrystal (1:1 M ratio) was successfully prepared by slow evaporation from both acetone and ethyl acetate. Acetone, ethyl acetate, and ethanol were all successfully used to prepare cocrystals by LAG and spray drying. The powders obtained were characterized by X-ray diffractometry (XRD), differential scanning calorimetry (DSC), thermogravimetry (TGA), and Fourier transform infrared spectroscopy (FTIR). Laser diffraction analysis indicated a median particle size (D50) for spray-dried powders prepared from acetone, ethanol, and ethyl acetate of 5.4 ± 0.7, 5.2 ± 0.1, and 5.1 ± 0.0 µm respectively, which are appropriate sizes for pulmonary delivery by means of a dry powder inhaler. The solubility of the CAF/DAP cocrystal in phosphate buffer pH 7.4, prepared by spray drying using acetone, was 506.5 ± 31.5 µg/mL, while pure crystalline DAP had a measured solubility of 217.1 ± 7.8 µg/mL. In vitro cytotoxicity studies using Calu-3 cells indicated that the cocrystals were not toxic at concentrations of 0.1 and of 1 mM of DAP, while an in vitro permeability study suggested caffeine may contribute to the permeation of DAP by hindering the efflux effect. The results obtained indicate that the CAF/DAP cocrystal, particularly when prepared by the spray drying method, represents a possible suitable approach for inhalation formulations with applications in pulmonary pathologies.

A promising oral fucoidan-based antithrombotic nanosystem: Development, activity and safety.

Fucoidan-loaded nanoparticles emerge as great candidates to oral anticoagulant therapy, due to increasing of bioavailability and circulation time of this natural anticoagulant. Crosslink between chitosan chains are performed using glutaraldehyde to confer higher gastric pH resistance to nanoparticle matrices. In this work, chitosan-fucoidan nanoparticles, without (NpCF) and with glutaraldehyde crosslink (NpCF 1% and NpCF 2%), were prepared to evaluate their anticoagulant, antithrombotic and hemorrhagic profile. Nanoparticles were characterized by average diameter, polydispersity index, zeta potential, Fourier transform infrared spectroscopy and fucoidan in vitro release. Anticoagulant and antithrombotic activities were determined by in vitro and in vivo models, respectively. Hemorrhagic profile was in vivo evaluated by tail bleeding assay. Preparations showed nanometric and homogeneous average diameters. Zeta potentials of NpCF and NpCF 1% were stable over gastrointestinal pH range, which was confirmed by low fucoidan release in gastric and enteric media. In pH 7.4, NpCF and NpCF 1% demonstrated fucoidan release of 65.5% and 60.6%, respectively, within the first 24 hours. In comparison to fucoidan, NpCF and NpCF 1% showed increased in vitro anticoagulant activity. A significant difference on oral antithrombotic profile of NpCF 1% was found in comparison to fucoidan. Bleeding profile of NpCF and NpCF 1% showed no differences to control group, indicating the safety of these systems. Surprisingly, oral antithrombotic profile of commercially available fucoidan, from Fucus vesiculosus, has not been previously determined, which reveals new possibilities. In this work, significant advances were observed in anticoagulant and antithrombotic profiles of fucoidan through the preparation of NpCF 1%.

Antileishmanial Thioureas: Synthesis, Biological Activity and in Silico Evaluations of New Promising Derivatives.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania. Currently, the drugs available for treatment of this disease present high toxicity, along with development of parasite resistance. In order to overcome these problems, efforts have been made to search for new and more effective leishmanicidal drugs. The aim of this study was to synthesize and investigate the leishmanicidal effect of N,N'-disubstituted thioureas against Leishmania amazonensis, with evaluation of their in silico pharmacokinetics and toxicity profiles. Our results showed that different thioureas could be obtained in high to moderate yields using simple reaction conditions. Nine thiourea derivatives (3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x and 3z) were active against parasite promastigotes (IC50 21.48-189.10 µM), with low cytotoxicity on mice peritoneal macrophages (CC50>200 µM), except for thiourea 3e (CC50=49.22 µM). After that, the most promising thioureas (3k, 3l, 3p, 3q and 3v) showed IC50 ranging from 70 to 150 µM against L. amazonensis amastigotes in infected macrophages. Except for thiourea 3p, the leishmanicidal activity of the derivatives were independent of nitric oxide (NO) production. Thioureas 3q and 3v affected promastigotes cell cycle without disturbing the mitochondrial membrane potential. Furthermore, our derivatives showed satisfactory theoretical absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. These data indicate that thiourea derivatives are good candidates as leading compounds for the development of new leishmanicidal drugs.

Targeted Nanosystems to Prostate Cancer.

Prostate cancer remains an increasingly common malignancy worldwide. Many advances in drug development have been achieved for the conventional treatments; however, chemotherapeutic agents are distributed nonspecifically in the body where they affect both prostate cancer and healthy cells. Limited dose achievable within the prostate tumor and suboptimal treatment due to excessive toxicities reveal the importance of the development of more specific mechanisms and ways of drug targeting to prostate tumor. In this context, nanotechnology, molecular biology and biochemistry have been applied in the pharmaceutical area for development of new targeted drug delivery nanosystems in order to improve its pharmacokinetic profile, raise the effectiveness of treatment; reduce side effects due to the preferential accumulation in prostate cancer cells, causing low concentrations in healthy tissues; and/or increase the drug chemical stability for improving the prostate cancer therapeutic. Thus, in this review, we will discuss the molecular and biochemical basis of prostate cancer as well as the advantages and disadvantages of conventional clinical treatments, different types and basic characteristics of nanosystems; how these systems can be targeted to prostate cancer, show successful patent examples of prostate cancer targeted nanosystems and present perspectives for the next 10-20 years in this area.

Development and Characterization of Nisin Nanoparticles as Potential Alternative for the Recurrent Vaginal Candidiasis Treatment.

The aim of this work was the development and characterization of nisin-loaded nanoparticles and the evaluation of its potential antifungal activity. Candidiasis is a fungal infection caused by Candida sp. considered as one of the major public health problem currently. The discovery of antifungal agents that present a reduced or null resistance of Candida sp. and the development of more efficient drug release mechanisms are necessary for the improvement of candidiasis treatment. Nisin, a bacteriocin commercially available for more than 50 years, exhibits antibacterial action in food products with potential antifungal activity. Among several alternatives used to modulate antifungal activity of bacteriocins, polymeric nanoparticles have received great attention due to an effective drug release control and reduction of therapeutic dose, besides the minimization of adverse effects by the preferential accumulation in specific tissues. The nisin nanoparticles were prepared by double emulsification and solvent evaporation methods. Nanoparticles were characterized by dynamic light scattering, zeta potential, Fourier transform infrared, X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. Antifungal activity was accessed by pour plate method and cell counting using Candida albicans strains. The in vitro release profile and in vitro permeation studies were performed using dialysis bag method and pig vaginal mucosa in Franz diffusion cell, respectively. The results revealed nisin nanoparticles (300 nm) with spherical shape and high loading efficiency (93.88 ± 3.26%). In vitro test results suggest a promising application of these nanosystems as a prophylactic agent in recurrent vulvovaginal candidiasis and other gynecological diseases.

In vitro and in vivo analysis of the antithrombotic and toxicological profile of new antiplatelets N-acylhydrazone derivatives and development of nanosystems: determination of novel NAH derivatives antiplatelet and nanotechnological approach.

BACKGROUND: Cardiovascular diseases are the most frequent cause of morbidity and mortality worldwide. Among the most important cardiovascular diseases are atherothrombosis and venous thromboembolism that present platelet aggregation as a key event. Currently, the commercial antiplatelet agents display several undesirable effects, which prompt the search for new compounds with better therapeutic index, more efficient body distribution and mechanism. METHODS: In this work we characterized in vivo and in vitro the antithrombotic and toxicological profiles of novel antiplatelet N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-carbohydrazides derivatives also comparing them with aspirin. In addition we also analyzed the stability of the more active compound after encapsulation in PLGA or PCL nanoparticles and the release profile of these new nanosystems. RESULTS: The biological results revealed not only the selective effect against arachidonic acid-induced platelet aggregation mainly for compounds 2c, 2e and 2h but also their in vivo active profile on thromboembolism pulmonary animal model with better survival rates (e.g. 82%) than aspirin (33%). The overall toxicological profile was determined by in vitro (MTT reduction tests, neutral red uptake in kidney VERO cells and hemolysis assays) and in vivo (pulmonary embolism) assays that pointed 2c as the most promising derivative with potential as a lead compound. By using the nanoprecipitation technique 2c was loaded into PLGA and PCL nanoparticles showing controlled release profile over 21days according to our drug release tests. CONCLUSION: According to our results compound 2c is the most interesting derivative for further studies as it showed the best activity and toxicological profile also allowing the nanoencapsulation process. Thus 2c may assist in determining a new potential therapy with favorable pharmacokinetics for treatment of thrombotic disorders.

Crystalline forms of nonprotein drugs filed in Brazil from 1995-2005.

In the present study, we evaluated 457 patent applications filed for crystalline forms of nonprotein drugs during the period 1995-2005 in Brazil. Online searches were conducted using the Instituto Nacional da Propriedade Industrial patent database and the Derwent Innovations Index(®). It was found that no patent applications in this area were filed by Brazilian applicants. It was also noted that only 61% of patent applications included the characterization of three or more crystalline phase techniques. In most applications, no determination of purity was provided for chemical and/or crystalline phases. In a subset of patent applications, those for blockbuster drugs, we found that approximately 90% did not meet Instituto Nacional da Propriedade Industrial acceptance criteria nor the criteria developed in this study. These results reveal that appropriate and thorough characterization of the crystalline forms of drugs is, indeed, lacking in patent applications.